1. Field of the Invention
This invention relates to methods of producing peptide-based anti-angiogenesis compounds using plasmin reductases, and specifically to methods of producing an A61 anti-angiogenic plasmin fragment using an annexin II heterotetramer. This invention also relates to compositions and methods useful for modulating angiogenesis.
2. Description of the Related Art
The following references listed below as part of this paragraph are cited throughout this disclosure using the associated numerical identifiers. Applicant makes no statement, inferred or direct, regarding the status of these references as prior art. These references are incorporated herein by reference:    1. O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Cao, Y., Moses, M., Lane, W. S., Sage, E. H., and Folkman, J. (1994) Cold Spring Harb. Symp. Quant. Biol. 59, 471-482    2. Dong, Z., Kumar, R., Yang, X., and Fidler, I. J. (1997) Cell 88, 801-810    3. Falcone, D. J., Khan, K. F., Layne, T., and Fernandes, L. (1998) J. Biol. Chem. 273, 31480-31485    4. Gately, S., Twardowski, P., Stack, M. S., Patrick, M., Boggio, L., Cundiff, D. L., Schnaper, H. W., Madison, L., Volpert, O., Bouck, N., Enghild, J., Kwaan, H. C., and Soff, G. A. (1996) Cancer Res. 56, 4887-4890    5. Gately, S., Twardowski, P., Stack, M. S., Cundiff, D. L., Grella, D., Castellino, F. J., Enghild, J., Kwaan, H. C., Lee, F., Kramer, R. A., Volpert, O., Bouck, N., and Soff, G. A. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10868-10872    6. Stathakis, P., Lay, A. J., Fitzgerald, M., Schlieker, C., Matthias, L. J., and Hogg, P. J. (1999) J. Biol. Chem. 274, 8910-8916    7. Patterson, B. C. and Sang, Q. A. (1997) J. Biol. Chem. 272,28823-28825    8. Cornelius, L. A., Nehring, L. C., Harding, E., Bolanowski, M., Welgus, H. G., Kobayashi, D. K., Pierce, R. A., and Shapiro, S. D. (1998) J. Immunol. 161, 6845-6852    9. Lijnen, H. R., Ugwu, F., Bini, A., and Collen, D. (1998) Biochemistry 37, 4699-4702    10. Morikawa, W., Yamamoto, K., Ishikawa, S., Takemoto, S., Ono, M., Fukushi, J., Naito, S., Nozaki, C., Iwanaga, S., and Kuwano, M. (2000) J. Biol. Chem.     11. Heidtmann, H. H., Nettelbeck, D. M., Mingels, A., Jager, R., Welker, H. G., and Kontennann, R. E. (1999) Br. J. Cancer 81, 1269-1273    12. O'Reilly, M. S., Wiederschain, D., Stetler-Stevenson, W. G., Folkman, J., and Moses, M. A. (1999) J. Biol. Chem. 274,29568-29571    13. Wu, H. L., Shi, G. Y., Wohl, R. C., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8793-8795    14. Kassam, G., Kwon, M., Yoon, C. -S., Graham, K. S., Young, M. K., Gluck, S., and Waisman, D. M. (2001) J. Biol. Chem. 276, 8924-8933    15. Stathakis, P., Fitzgerald, M., Matthias, L. J., Chesterman, C. N., and Hogg, P. J. (1997) J. Biol. Chem. 272, 20641-20645    16. Lay, A. J., Jiang, X. M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869-873    17. Kassam, G., Choi, K. S., Ghuman, J., Kang, H. M., Fitzpatrick, S. L., Zackson, T., Zackson, S., Toba, M., Shinomiya, A., and Waisman, D. M. (1998) J. Biol. Chem. 273, 4790-    18. Kassam, G., Le, B. H., Choi, K. S., Kang, H. M., Fitzpatrick, S. L., Louie, P., and Waisman, D. M. (1998) Biochemistry 37, 16958-16966    19. Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. -M., Fogg. D. K., and Waisman, D. M. (2000) Biochemistry 39, 1021-1028    20. Kang, H. M., Choi, K. S., Kassam, G., Fitzpatrick, S. L., Kwon, M., and Waisman, D. M. (1999) Trends. Cardiovasc. Med. 9, 92-102    21. Khanna, N. C., Heiwig, E. D., Ikebuchi, N. W., Fitzpatrick, S., Bajwa, R., and Waisman, D. M. (1990) Biochemistry 29, 4852-4862    22. Filipenko, N. R. and Waisman, D. M. (2001) J. Biol. Chem. 276, 5310-5315    23. Ayala-Sanmartin, J., Vincent, M., Sopkova, J., and Gallay, J. (2000) Biochemistry 39, 15179-15189    24. Ghahary, A., Tredget, E. E., Chang, L. J., Scott, P. G., and Shen, Q. (1998) J Invest Dermatol. 110, 800-805    25. Johnsson, N. and Weber, K. (1990) J. Biol. Chem. 265, 14464-14468    26. Mai, J., Waisman, D. M., and Sloane, B. F. (2000) Biochim. Biophys. Acta 1477,215-230    27. Rubartelli, A., Bajetto, A., Allavena, G., Wolhman, E., and Sitia, R. (1992) J. Biol. Chem. 267, 24161-24164    28. Terada, K., Manchikalapudi, P., Noiva, R., Jauregui, H. O., Stockert, R. J., and Schilsky, M. L. (1995) J. Biol. Chem. 270, 20410-20416    29. Soderberg, A., Sahaf, B., and Rosen, A. (2000) Cancer Res. 60, 2281-2289    30. Heuck, A. P. and Wolosiuk, R. A. (1997) J. Biochem. Biophys. Methods 34, 213-225    31. Heuck, A. P. and Wolosiuk, R. A. (1997) Anal. Biochem. 248, 94-101    32. Lahav, J., Gofer-Dadosh, N., Luboshitz, J., Hess, O., and Shaklai, M. (2000) FEBS Lett. 475, 89-92    33. Langenbach, K. J. and Sottile, J. (1999) J. Biol. Chem. 274, 7032-7038    34. Essex, D. W. and Li, M. (1999) Br. J. Haematol. 104, 448-454    35. Mayadas, T. N. and Wagner, D. D. (1992) Proc. Natl. Acad. Sci. U.S.A 89, 3531-3535    36. O'Neill, S., Robinson, A., Deering, A., Ryan, M., Fitzgerald, D. J., and Moran, N. (2000) J. Biol. Chem. 275, 36984-36990
Annexin II heterotetramer (“AIIt”) is a Ca2+-binding protein complex that binds tPA, plasminogen and plasmin and stimulates both the formation and autoproteolysis of plasmin at the cell surface (17-19) (reviewed in (20)). The protein consists of two copies of an annexin II 36 kDa subunit (p36) called annexin II and two copies of an 11 kDa subunit (p11) called S100A10. It is known in the art that the carboxyl-terminal lysines of the p11 subunit plays a key role in plasminogen binding and activation (18).
Angiostatin was originally identified in the urine of mice bearing Lewis lung carcinoma (LLC) as a 38 kDa proteolytically-derived fiagment of plasminogen which encompassed the first four kringle domains of plasminogen (Lys78-Ala440 according to SEQ ID NO:1). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases. Angiostatin was also characterized as a specific antiangiogenic protein that blocked microvascular endothelial cell proliferation but not the proliferation of nonendothelial cells (1).
Angiostatin is a member of a family of antangiogenic plasminogen fragments (“AAPFs”). Physiologically relevant AAPFs include a 38 kDa AAPF isolated from the conditioned media of tumor-infiltrating mnacrophages (2), a 43 kDa and 38 kDa AAPF identified in the conditioned media of Chinese hamster ovary and HT1080 fibrosarcoma cells and a 48 kDa AAPF present in macrophage conditioned media (3). Other AAPFs include a 43 kDa and a 38 kDa AAPF isolated from the conditioned media of human prostrate carcinoma PC-3 cells (4; 5) and AAPFs of 66, 60 and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (6). Since the carboxyl-terminus of most of these AAPFs was not determined, the exact primary sequence of most of the AAPFs is unknown.
Two distinct pathways have been identified for the formation of AAPFs. First, certain proteinases can directly cleave plasminogen into AAPFs. These proteinases include metalloelastase, gelatinase B (MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), cathepsin D and prostate-specific antigen (7-11). The source of these proteinases may be tumor-infiltrating macrophages (2) or the cancer cells themselves. For example, the conversion of plasminogen to angiostatin by macrophages is dependent on the release of metalloelastase from these cells. In comparison, Lewis lung carcinoma cells release MMP-2 which also cleaves plasminogen to angiostatin (12). Second, AAPFs are also generated by a three step mechanism which involves the conversion of plasminogen to plasmin by urokinase-type plasminogen activator (“uPA”), the autoproteolytic cleavage of plasmin and the release of the resultant plasmin fragment by cleavage of disulfide bonds. The cleavage of the plasmin disulfide bonds can be accomplished by free sulthydryl group donors (FSD) such as glutathione or by hydroxyl ions at alkaline pH (4; 5; 13; 14). Alternatively, the plasmin disulfide bonds can be cleaved enzymatically by a plasmin reductase such as phosphoglycerate kinase (15; 16).
In co-pending patent application PCT/US01/44515 (published as WO0244328 A and reference 14), which is incorporated herein by reference, it was shown that the primary AAPF present in mouse and human blood has a molecular weight of 61 kDa. This AAPF, called A61, was produced in a cell-free system consisting of uPA and plasminogen. A, was shown to be a novel four-kringle containing plasminogen fragment consisting of the amino acid sequence, Lys78-Lys468 (SEQ ID NO:1) (14). The release of A61 from plasmin required cleavage of the Lys468-Gly469 (SEQ ID NO:1) bond by plasmin autoproteolysis and also cleavage of the Cys462-Cys541 (SEQ ID NO:1) disulfide. Since A61 was generated in a cell-free system from plasmin at alkaline pH in the absence of sulfhydryl donors, it was concluded that cleavage of the Cys462-Cys541 disulfide was catalyzed by hydroxyl ions in vitro. In contrast, at physiological pH, it was observed that the conversion of plasminogen to Al was very slow. These results contrasted with the observation that at physiological pH, HT1080 fibrosarcoma and bovine capillary endothelial (BCE) cells stimulated the rapid formation of A61. Heretofore, the mechanism by which these cells stimulated plasmin reduction and the release of A61 from plasmin was unclear.